CD8 expressing T cells (TCD8+) play a critical role in eradicating intracellular parasites such as viruses. TCD8+ recognize MHC class I molecules in a complex with peptides of 8 to 10 residues derived from viral proteins located in the cytosol. There is tremendous interest in the mechanism by which peptides are generated. Indirect evidence implicates proteasomes in the generation of antigenic peptides. Proteasomes are abundant macromolecular structures present in the cytosol and nucleus in cells, and have multiple protease activities. They are thought to be responsible for energy dependent proteolysis in which ubiquitin plays a prominent role in targeting proteins for destruction. Although it is believed that at least some proteolysis occurs in the cytosol, it is uncertain whether the ultimate determinants are generated in the cytosol or whether additional trimming occurs following transport from the cytosol. We have followed several approaches to assess the site and nature of proteolytic mechanisms utilized in the generation of antigenic peptides. First, we have examined the relationship between metabolic stability of a protein and its efficiency as a source of antigenic peptides. Second, we tested the effects of proteasome inhibitors on the generation of class I binding peptides. Our findings in these areas suggest that a major source of antigenic peptides is a pool of defective polypeptides that are destined for degradation, and that proteasomes are not the only cytosolic proteases involved in the production of antigenic peptides.